Method of diagnosing renal dysfunction in a cat based on non-cauxin protein levels

ABSTRACT

Disclosed is a method of diagnosing renal dysfunction in a cat by removing cauxin from cat urine and detecting a protein in the cat urine sample from which cauxin has been removed. Cauxin is removed from cat urine by bringing the cat urine into contact with a lectin or with an anti-cauxin antibody that specifically binds to cauxin. Cauxin may be removed using a column filled with a carrier that is bound to the lectin or to the anti-cauxin antibody. The lectin may be  Lens culinaris  lectin. Detection of the cat urinary protein from which cauxin has been removed is performed with a urine test strip.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.12/452,687, which is a National Stage of International PatentApplication No. PCT/JP2007/064724, filed Jul. 20, 2007. The disclosuresof application Ser. Nos. 12/452,687 and PCT/JP2007/064724 are expresslyincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to a urine test for cats in the veterinaryclinical field, a method for diagnosing renal dysfunction in cats bydetecting a cat urinary protein, and a diagnostic agent for renaldysfunction in cats.

BACKGROUND ART

As a cat kept as a pet becomes older, it becomes more subject to kidneydiseases. Since the diseases occupy a higher rank of the cause of deathof cats, early diagnosis for kidney diseases in cats has become animportant object in the clinical settings of the veterinary clinicalfield for small animals. A measurement of a protein quantity excreted inurine has been used as an initial diagnosis for kidney diseases inhumans. Proteinuria has been utilized as a diagnostic marker to indicatea renal abnormality at an early stage. However, in cats, physiologicalproteinuria (cauxin urine) and diseased proteinuria cannot bedistinguished with a commercially available urine test strip because ahigh level of proteinaceous cauxin derived from the kidney of evenhealthy cats exists in the urine. A method for detecting kidney diseasesin cats by measuring urinary cauxin has been reported, but the methodrequires a special reagent (cf. Patent Document 1). Therefore, a teststrip for protein measurement generally used in the urinalysis cannot beused, and development of a simple and easy analysis method is desired.In addition, since there is a big sex difference in the content ofcauxin present in the urine, it has been difficult in this respect todetect kidney diseases by the detection of cauxin.

-   Patent Document 1: Japanese Patent Application Laid-Open No.    2003-250575

SUMMARY OF THE INVENTION

The present invention is intended to provide a method for diagnosingkidney diseases in cats by detecting a cat urinary protein without beingaffected by cauxin, using a test strip for protein measurement generallyused in the urinalysis, as well as to provide a diagnostic agenttherefor.

Cauxin secreted in the urine of healthy cats is a glycoprotein having amolecular weight of approximately 70 kd. The present inventors analyzedthe binding between a lectin which is a plant seed-derived protein thatspecifically binds to glycoproteins, and a cat urinary protein, andfound that a lectin derived from Lens culinaris specifically binds tocauxin, but binds to other urinary proteins to a lesser extent.Accordingly, a gel which had been bound to lentil lectin (lentil lectinSepharose) was added to a cat urine sample and suspended, and thenanalysis of proteins in the supernatant was performed. As a result, onlycauxin was adsorbed on the gel, and the other urinary proteins werecollected in the supernatant. Use of this method makes it possible tospecifically remove only cauxin from the cat urine and easily detect theurinary protein derived from renal dysfunction. The present inventionhas been completed based on these findings.

In other words, aspects of the present invention are as follows.

[1] A method of detecting a cat urinary protein derived from renaldysfunction in a cat, including removing cauxin from cat urine anddetecting a protein in a cat urine sample from which cauxin has beenremoved.

[2] The method of detecting a cat urinary protein derived from renaldysfunction in a cat according to item [1], wherein cauxin is removedfrom the cat urine by bringing the cat urine into contact with a lectinor an anti-cauxin antibody that specifically binds to cauxin.[3] The method of detecting a cat urinary protein derived from renaldysfunction in a cat according to item [2], wherein cauxin is removedfrom the cat urine by using a carrier which is bound to a lectin or ananti-cauxin antibody that specifically binds to cauxin.[4] The method of detecting a cat urinary protein derived from renaldysfunction in a cat according to item [3], wherein cauxin is removedfrom the cat urine by using a column filled with a carrier which isbound to a lectin or an anti-cauxin antibody that specifically binds tocauxin.[5] The method of detecting a cat urinary protein derived from renaldysfunction in a cat according to any one of items [2] to [4], whereinthe lectin is Lens culinaris lectin.[6] The method of detecting a cat urinary protein derived from renaldysfunction in a cat according to any one of items [1] to [5], whereinthe detection of the cat urinary protein from which cauxin has beenremoved is performed with a urine test strip for the detection of aurinary protein.[7] The method of detecting a cat urinary protein derived from renaldysfunction in a cat according to any one of items [2] to [6], whereinthe lectin that specifically binds to cauxin is Lens culinaris lectin.[8] The method of detecting a cat urinary protein derived from renaldysfunction in a cat according to any one of items [3] to [7], whereinthe carrier which is bound to the lectin that specifically binds tocauxin is Sepharose (registered trademark) or TOYOPEARL (registeredtrademark).[9] A diagnostic method of renal dysfunction in a cat, includingdetecting a cat urinary protein by the detection method of a cat urinaryprotein according to any one of items [1] to [8] and deciding whether ornot the cat suffers from renal dysfunction.[10] A detection reagent for a urinary protein derived from renaldysfunction in a cat, including a carrier which is bound to a lectin oran anti-cauxin antibody that specifically binds to cauxin, and a urinetest strip for the detection of a urinary protein.[11] The detection reagent for a urinary protein derived from renaldysfunction in a cat according to item [10], including a column filledwith a carrier which is bound to a lectin or an anti-cauxin antibodythat specifically binds to cauxin, and a urine test strip for thedetection of a urinary protein.[12] The detection reagent for a urinary protein derived from renaldysfunction in a cat according to item [10] or [11], wherein the lectinthat specifically binds to cauxin is Lens culinaris lectin.[13] The detection reagent for a urinary protein derived from renaldysfunction in a cat according to any one of items [10] to [12], whereinthe carrier which is bound to the lectin that specifically binds tocauxin is Sepharose (registered trademark) or TOYOPEARL (registeredtrademark).[14] A diagnostic agent for renal dysfunction in a cat, including thedetection reagent for a urinary protein derived from renal dysfunctionin a cat according to any one of items [11] to [13].

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a diagnostic kit for a urinaryprotein of cats.

FIG. 2 is a diagram showing a reaction of various lectins with cauxin bya lectin blot analysis.

FIG. 3 is a diagram showing the results of the removal of cauxin from aurine sample by LCA-cauxin.

FIG. 4 is a diagram showing a production method and a use method of acarrier column utilizing a blue chip.

FIG. 5 is a diagram showing the results of SDS-PAGE of a urine samplewhich has been passed through a carrier column.

FIG. 6A is a graph showing the amount of proteins in a urine samplewhich has been passed through a carrier column (LCA-Sepharose column).

FIG. 6B is a graph showing the amount of proteins in a urine samplewhich has been passed through a Sepharose CL-6B column used as acontrol.

FIG. 7 is a diagram showing the results of SDS-PAGE of a urine samplefrom which cauxin has been removed using a TOYOPEARL carrier.

FIG. 8A is a graph showing the results of the quantification of theamount of cauxin by the Bradford quantification method using an SDS-PAGEgel of a urine sample from which cauxin has been removed using aTOYOPEARL carrier.

FIG. 8B is a graph showing the results of the quantification of theamount of cauxin by densitometric quantification using an SDS-PAGE gelof a urine sample from which cauxin has been removed using a TOYOPEARLcarrier.

FIG. 9 is a diagram showing the results of SDS-PAGE of a urine samplefrom which cauxin has been removed using a cross-linked TOTOPEARLcarrier.

FIG. 10A is a graph showing the results of the quantification of theamount of cauxin by the Bradford quantification method using an SDS-PAGEgel of a urine sample from which cauxin has been removed using across-linked TOYOPEARL carrier.

FIG. 10B is a graph showing the results of the quantification of theamount of cauxin by densitometric quantification using an SDS-PAGE gelof a urine sample from which cauxin has been removed using across-linked TOYOPEARL carrier.

FIG. 11 is a diagram showing the results of the measurement of theamount of urinary proteins in a urine sample which has been subjected toa treatment for the removal of cauxin and a urine sample which has notbeen subjected to a treatment for the removal of cauxin, using a urinetest strip (PRETEST, Wako Pure Chemical Industries, Ltd.).

FIG. 12 is a photograph showing the results of SDA-PAGE of the urineobtained by adding, to a cross-linked TOYOPEARL carrier, cat urine towhich serum has been added, and passing the urine through the carrier.

FIG. 13 is a graph showing the results of the quantification by theBradford method of the urine obtained by adding, to a cross-linkedTOYOPEARL carrier, cat urine to which serum has been added, and passingthe urine through the carrier.

FIG. 14 is a photograph showing the results of SDS-PAGE of the urineobtained by adding, to a cross-linked TOYOPEARL carrier, urine of a catsuffering from a kidney disease, and passing the urine through thecarrier.

FIG. 15 is a photograph showing the results of SDS-PAGE of the urineobtained by adding, to a cross-linked TOYOPEARL carrier, urine of a catsuffering from a kidney disease, and passing the urine through thecarrier.

FIG. 16 is a photograph showing the results of SDS-PAGE of the urineobtained by adding, to a cross-linked TOYOPEARL carrier, urine of a catsuffering from a kidney disease, and passing the urine through thecarrier.

BEST MODES FOR CARRYING OUT THE INVENTION

In the method of the present invention, renal dysfunction in cats isdiagnosed by removing cauxin from cat urine and measuring proteins thatremaining in the cat urine.

The cat in the present invention means an animal belonging to the familyFelidae and examples there of include animals belonging to the subfamilyFelidae, such as Felis catus (domestic cat), Puma concolor (puma), andFelis silvestris (wild cat), animals belonging to the subfamilyPantherinae, such as leopard, lion, tiger, and jaguar, and animalsbelonging to the subfamily Acinonychinae, such as cheetah.

Removal of cauxin from cat urine can be performed using a substance thatspecifically binds to cauxin. Here, the substance that specificallybinds to cauxin means a substance that specifically binds to cauxinpresent in cat urine, but does not bind to a protein other than cauxinpresent in cat urine, particularly a protein present in the urine at ahigh concentration in the case where the cat suffers from renaldysfunction. Examples of the protein other than cauxin present in thecat urine include albumin, globulin, β2-microglobulin, and the like.Examples of the substance that specifically binds to cauxin include alectin, an anti-cauxin antibody, and the like. Examples of the lectinthat specifically binds to cauxin include concanavalin A (Con A), Lensculinaris (lentil) lectin (LCA), Arachis hypogaea (peanut) lectin (PNA),Ricinus communis (castor bean) lectin (RCA), Phaseolus vulgaris (kidneybean or common bean) lectin, Triticum vulgare (wheat germ) lectin (WGA),Pisum sativum (pea) lectin (PSA), Vicia faba (broad bean) lectin (VFA),and the like. Among them, Lens culinaris lectin (LCA) having a highbinding affinity to cauxin is preferable. Moreover, compounds thatspecifically bind to cauxin, such as substrates for cauxin and analogsthereof, can be mentioned.

An anti-cauxin antibody is easily available as a polyclonal ormonoclonal antibody by purifying cauxin from cat urine and immunizing ananimal with it.

Removal of cauxin may be performed by bringing cat urine into contactwith a substance that specifically binds to cauxin and removing theresulting complex of cauxin with the substance that specifically bindsto cauxin. For example, cat urine from which cauxin has been removed canbe obtained by allowing a substance that specifically binds to cauxin tobind to a suitable carrier, packing the bound carrier into a column,passing cat urine through the column, and collecting the passed urine.In addition, the complex of the carrier with cauxin may be removed byadding, to cat urine, a carrier to which a substance that specificallybinds to cauxin is bound, and performing a reaction for a fixed time,followed by centrifugation or filtration. Furthermore, the complex ofcauxin with a substance that specifically binds to cauxin may be removedby adding, to urine, a substance in a free state that specifically bindsto cauxin, and performing a reaction for a fixed time, followed bycentrifugation. As the carrier for binding the substance thatspecifically binds to cauxin, celluloses, such as Sepharose (registeredtrademark), Sephadex (registered trademark), Cellulofine (registeredtrademark), and TOYOPEARL (registered trademark) for affinitychromatography (AFC type), and resins, such as agarose, dextran, silica,a vinyl polymer, and a synthetic polymer can be used, and the substanceand cauxin can be bound together by a known method. For example,TOYOPEARL AF-Tresyl-650, TOYOPEARL AF-Carboxy-650, TOYOPEARLAF-Formyl-650, TOYOPEARL AF-Amino-650, and TOYOPEARL AF-Epoxy-650 (TosohCorporation) can be used. In addition, carriers to which various lectinsare bound are commercially available, and these commercially availablelectin-bound carriers may be used. Examples of the commerciallyavailable carrier include LCA-Sepharose (Amersham plc), Lens culinarislectin-agarose (Seikagaku Corporation), and the like. The method forseparating a protein that binds to a lectin by utilization of a lectinis known as affinity chromatography using a lectin column, anddetermination of lectins to be used, binding between a lectin and acarrier, preparation of a column, and determination of bindingconditions between a lectin column and proteins, and the like, can beperformed according to the description, for example, in “ExperimentCourses in New Biochemistry 3 (Shin Seikagaku Jikken Koza 3),Carbohydrate I, Glycoproteins (the First Volume), pages 3 to 29, TOKYOKAGAKU DOJIN CO., LTD, published on May 21, 1990.”

Moreover, when a column containing a carrier to which a substance thatspecifically binds to cauxin is bound is used, a minicolumn ispreferably used so that the detection of the urinary protein is possibleeven if a small amount of cat urine is used. The capacity of theminicolumn is 100 μL to 2000 μL, for example. For example, cat urinefrom which cauxin has been removed can be obtained by stuffing absorbentcotton, filter paper, or the like on the apex of a commerciallyavailable tip (blue tip or yellow tip) for micropipettes made of a resinsuch as polypropylene, further stuffing a carrier to which a substancethat specifically binds to cauxin is bound, thereby to prepare aminicolumn for the removal of cauxin, adding the cat urine to theminicolumn, and collecting the urine which has passed through thecolumn. In this case, a buffer such as a Tris buffer or a phosphatebuffer may be used for the equilibration of the column and for thepassage of the protein adsorbed on the column.

On this occasion, the quantitative rate of the substance thatspecifically binds to cat urine and cauxin is not limited, and thesubstance may be brought into contact with the cat urine at aquantitative rate being able to remove all the cauxin in the cat urine.Cauxin exists in about 0.9 mg/mL in healthy cat urine, and in about 0.1mg/mL or less in the urine of a cat suffering from renal dysfunction. Inthe present invention, a treatment for the removal of cauxin is carriedout so that 90% or more, and preferably 95% or more of cauxin present inhealthy cat urine is removed.

In order to detect renal dysfunction in a cat, proteins present in thecat urine from which cauxin has been removed may be measured. Examplesof the protein able to serve as a marker of renal dysfunction includealbumin, lysozyme, and haptoglobin.

The measurement of a urinary protein can be performed using a test stripfor urinary protein measurement.

As such a test strip for urinary protein measurement, any knowncommercially available one may be used. Examples of the commerciallyavailable test strip include PRETEST manufactured by Wako Pure ChemicalIndustries Ltd. The test strip is a test strip which is soaked with a pHindicator such as bromophenol blue or tetrabromophenol blue and a pHbuffer solution such as a citrate buffer solution, and detects proteinsbased on a protein error of the pH indicator.

In addition, such detection of proteins can be performed by the Bradfordmethod, the BCA protein assay, the sulfosalicylic acid method, and thelike.

In the case of a cat, it is possible to diagnose whether or not the catsuffers from renal dysfunction by the protein concentration in the urinefrom which cauxin has been removed. If the protein concentration in thecat urine is about 100 to several hundred μg/mL preferably about 100 toabout 200 μg/mL or more, it is very likely that the cat suffers fromrenal dysfunction. Whether or not the cat suffers from renal dysfunctionis finally diagnosed by the general judgment of a veterinarian.

According to the method of the present invention, the protein in the caturine derived from renal dysfunction can be detected and renaldysfunction in a cat can be diagnosed.

Further, the present invention includes a carrier which is bound to asubstance that specifically binds to cauxin or a column filled with thecarrier, as well as a reagent or a kit for the detection of a caturinary protein derived from renal dysfunction including a test stripfor the detection of a urinary protein. In addition, the presentinvention includes a diagnostic agent or a diagnostic kit for renaldysfunction in cats, including the reagent.

Hereinafter, the present invention will be specifically explained byreferring to the following examples, but the scope of the presentinvention should not be limited thereto.

Example 1

A lectin is a protein having an activity to recognize a sugar chain of aglycoprotein and specifically bind to it. As a result of studies on thebinding activity of various lectins to the sugar chain of cauxin, itbecame clear that Lens culinaris lectin (hereinafter abbreviated as LCA)showed a high affinity to urinary cauxin and was specifically bound tothe urinary cauxin (FIG. 2).

An experiment for removing urinary cauxin was performed by adding caturine to LCA-Sepharose (Amersham) which is an affinity carrier bound toLCA. A urine sample (50 μL) was added to 100 μL of LCA-Sepharose, themixture was tumbled and mixed, allowed to stand on ice for 5 minutes,and centrifuged to collect a supernatant. Using as a control a Sepharosecarrier which was not bound to LCA, a similar experiment was carriedout. Each sample was subjected to SDS-PAGE under non-reductiveconditions and CBB staining was performed to detect proteins (FIG. 3).

As a result, it was revealed that the amount of the urinary protein(albumin) except cauxin hardly changed, but cauxin was specificallyadsorbed on the LCA-Sepharose carrier and was removed from the sample.

Example 2 Reaction of Lectin Carrier with Urine Utilizing Blue Tip forMicropipettes

As shown in FIG. 4, a blue tip was filled with 200 μL of LCA-Sepharoseand equilibrated with TBS, and 300 μL of urine was applied thereto topass through the column, and drops of the passed urine was collected(one drop: about 50 μL). The collected urine (10 μL) was applied toSDS-PAGE under non-reductive conditions and the gel was stained with CBB(FIG. 5), and then proteins were quantified by the Bradford method (FIG.6).

In the control, the equilibrated buffer was all replaced by the urine atthe stage of the third to fourth drops. It was elucidated that, on thetip stuffed with 200 μL of LCA Sepharose, 90% or more of proteins wereadsorbed in the third drop fraction. It is believed that it becomespossible to obtain a fraction from which cauxin is almost wholly removedby adding urine to such a minicolumn, and collecting a sample afterapplication of several drops of the urine.

Example 3 Removal of Cauxin by Using TOYOPEARL Carrier

An experiment similar to Example 2 was performed using TOYOPEARLAF-Tresyl 650M (TOHSOH, hereinafter abbreviated as TOYOPEARL) as anaffinity carrier capable of binding to LCA. TOYOPEARL was weighed in anamount of 0.1 g (about 400 μL), and LCA which had been dissolved in a0.1 M sodium phosphate buffer (pH 7.4, 2 mL) was added thereto so as toattain a ratio of 15 mg/mL (carrier), and a reaction was performedovernight by tumbling mixing at 4° C. Thereafter, the carrier was washedwith 5-fold amount of 1 M NaCl, and blocking was performed with 0.1 MTris-HCl+0.5 M NaCl at room temperature for 1 hour. The collected urinesample was subjected to SDS-PAGE, and a band of cauxin was quantifiedfrom an image of the gel stained with CBB (FIG. 7), using an imageanalysis program (BIO-RAD, Molecular Analyst) (FIGS. 8A and 8B). FIG. 8Ashows the results obtained by the Bradford quantification method, andFIG. 8B shows the results obtained by the densitometric quantificationmethod. As a result, compared to the control carrier (only a blockingtreatment was performed without addition of LCA), a urine sample of thesecond to eighth drops from which 90 to 95% of cauxin had been removedwas obtained. When the total protein concentration of the urine afterbeing passed through the carrier was measured by a dye-binding method(BIO-RAD Quick Start Bradford Dye Reagent), the protein concentrationwas reduced to 25 to 30% of that before column passage. By thisoperation, a two stage change is observed in the judgment of a generalurine test strip (PRETEST: Wako Pure Chemical Industries, Ltd.). Fromthis result, a technique using an LCA column is considered to beeffective for removing cauxin in cat urine.

A similar experiment was carried out twice. In the second experiment, 5mm at the apex of a blue tip was cut, and the above-mentioned carrier(200 μL) was filled into the blue tip whose apex had been stuffed withabsorbent cotton and then equilibrated with TBS (Tris-Buffered Saline).Urine (500 μL) was applied to this column, and eight fractions of twodrops each (about 60 μL) were collected from the urine which had passedthe column. Protein quantification of each of the collected fractionswas performed by the Bradford method, and 10 μL of the fraction wereapplied to SDS-PAGE (12% acrylamide gel) under non-reductive conditions,and then the gel was stained with CBB. After destaining, a band ofcauxin was quantified by densitometry. A carrier wherein the reactivegroup was blocked with 0.2 M Tris-HCl (pH 7.4) without addition of alectin was filled into the same tip in the same amount, and this wasused as a control.

FIG. 9 shows the results of SDS-PAGE; FIG. 10A shows the results of theBradford assay; and FIG. 10B shows the results of the quantificationusing densitometry. In the protein quantification by the Bradfordmethod, about 88% of total proteins in the fifth fraction wherein TBShad been all replaced by urine was adsorbed on the carrier, and, in thequantification of a band of cauxin by densitometry, about 96% of cauxinwas adsorbed on the carrier. Thus, it was confirmed that cauxin can beremoved from cat urine.

Using a urine sample from which cauxin had been removed by the methodmentioned above and a urine sample from which cauxin had not beenremoved, a urinary protein was measured using a urine test strip(PRETEST: Wako Pure Chemical Industries, Ltd.). The results are shown inFIG. 11. As shown in FIG. 11, the degree of the color development wasdecreased in the urine sample from which cauxin had been removed.

When a similar study was performed by using TOYOPEARL AF-Formyl 650Minstead of the above-mentioned TOYOPEARL AF-Tresyl 650M, similar resultswere obtained.

Example 4 Influences of Serum Protein

Using the column prepared in Example 3, influences of serum protein inthe removal of cauxin were analyzed. Cat serum (10 μL) was added to 490μL of healthy male cat urine, and the mixture was added to a carriercolumn, and the urinary protein which had passed the column was analyzedby the method mentioned above. FIG. 12 shows the results of SDS-PAGE ofeach fraction, and FIG. 13 shows the protein concentration of eachfraction as measured by the Bradford method. As shown in the drawings,it was proved that only cauxin is selectively adsorbed on the columneven if a high level of a serum protein exists.

Example 5 Measurement Using Urine of Cat Suffering from RenalDysfunction

An experiment similar to Examples 3 and 4 was performed using 500 μL ofurine, in which almost no cauxin was found, of three cats with a kidneydisease, and an elution pattern of proteins was compared to that of thecontrol by SDS-PAGE of each fraction of the fifth to sixth fractions.The results of SDS-PAGE of each cat urine are shown in FIGS. 14 to 16.As shown in the drawings, in three cat urine samples, the patterns ofthe urinary protein were different from each other, but it wasunderstood that almost no binding of proteins other than cauxin found incats with a kidney disease to the LCA column is found.

INDUSTRIAL APPLICABILITY

In the urinalysis for cats using a protein test strip used in usualurinalyses, it is difficult to make a distinction between cauxin presentin healthy urine in large quantities and a urinary protein derived fromkidney diseases (renal dysfunction), but a measurement using the presenttechnique makes it possible to selectively detect the latter proteinonly.

All publications, patents, and patent applications cited in thisapplication are intended to be incorporated herein by reference in theirentireties.

What is claimed is:
 1. A method of determining whether a cat has renal dysfunction, the method comprising removing cauxin from a urine sample from the cat; measuring a level of protein in the urine sample from which cauxin has been removed; and determining whether or not the cat has renal dysfunction using the measured level of protein.
 2. The method according to claim 1, wherein cauxin is removed from the urine sample by bringing the urine sample into contact with a lectin that specifically binds to cauxin or an anti-cauxin antibody that specifically binds to cauxin.
 3. The method according to claim 2, wherein the lectin that specifically binds to cauxin is Lens culinaris lectin.
 4. The method according to claim 1, wherein cauxin is removed from the urine sample by using a carrier, the carrier being bound to a lectin that specifically binds to cauxin or the carrier being bound to an anti-cauxin antibody that specifically binds to cauxin.
 5. The method according to claim 4, wherein the lectin that specifically binds to cauxin is Lens culinaris lectin.
 6. The method according to claim 4, wherein the carrier comprises agarose or vinyl polymer.
 7. The method according to claim 4, wherein cauxin is removed from the urine sample by using a column filled with the carrier.
 8. The method according to claim 7, wherein the lectin that specifically binds to cauxin is Lens culinaris lectin.
 9. The method according to claim 7, wherein the carrier comprises agarose or vinyl polymer.
 10. The method of according to claim 2, wherein the level of protein is measured using a urine test strip. 